KR20190026925A - Desaturized antibodies specifically binding to clec14a and uses thereof - Google Patents

Abstract

The present invention relates to a desaturase antibody that specifically binds to C-type lectin domain family 14, member A (clec14a), and uses thereof. More specifically, the present invention relates to a desaturase antibody comprising a light chain variable region comprising a CDR1 of a specific sequence and specifically binding to clec14, and its use, for example, for the prevention or treatment of an angiogenesis- ≪ / RTI >

Description

Desaturized antibodies specifically binding to clec14a and uses thereof

The present invention relates to a desaturase antibody that specifically binds to C-type lectin domain family 14, member A (clec14a), and uses thereof. More specifically, the present invention relates to a desaturase antibody comprising a light chain variable region comprising a CDR1 of a specific sequence and specifically binding to clec14, and its use, for example, for the prevention or treatment of an angiogenesis- ≪ / RTI >

Tumor angiogenesis plays an important role in tumor progression. Vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) are important factors in angiogenesis and angiogenesis is a very promising target for cancer treatment. The anti-VEGF antibody anti-VEGF antibody bevacizumab (avastin) is useful for the treatment of metastatic colorectal cancer, renal cell carcinoma, non-small cell lung cancer, And malignant brain glioma disease. ≪ Desc / Clms Page number 2 > Cetuximab, an anti-EGFR antibody, inhibits endothelial cell contact and is involved in the formation of blood vessels such as VFGF, interleukin-8 and basic fibroblast growth factor The expression of the neoplastic factor can be suppressed.

However, since avastin is not a clinically effective drug in a single preparation, it is currently being used in combination therapy with various chemical agents. In the case of combination therapy, a variety of chemical agents are used in combination with treatment, There is a risk of causing side effects. In addition, Avastin inhibits VEGF signaling in normal vessels as well as tumor vessels, leading to side effects such as proteinuria, hypertension, bleeding, and gastrointestinal perforation by inducing normal vascular defects It is known.

Furthermore, high-level VEGF-A, -B, -C, PIGF (placental growth factor), VEGF receptor-1 (VEGF receptor-1 ), And the expression of various pro-angiogenic soluble factors and receptors may be increased even when the VEGF neutralizing antibody is treated.

There is a need to develop a new antibody drug that will improve the side effects and tolerance of Avastin. In this regard, the inventors of the present application have discovered that an extracellular domain that constitutes a series of epidermal growth factor-like domains, the C-type lectin-like domain (CTLD) and the sushi-like domain We found that CTLD of Clec14a, a type I transmembrane protein, plays an important role in actin cytoskeletal rearrangement, which is important for cell migration. Based on this, clec14a-CTLD-specific human antibodies were screened, and the selected antibodies had high cross-reactivity to human and mouse clec14a-CTLD, inhibited the migration of vascular endothelial cells, inhibited tube formation, It has been confirmed that inhibition of cell contact can inhibit tumor angiogenesis and has been filed in International Publication No. WO2013187556.

However, in order to evaluate the efficacy and toxicity of developed antibodies in animal models, large-scale production of antibodies is required and the stability of the antibody itself is important. However, in the case of clec14a-CTLD-specific human antibodies, protein aggregation occurs during purification It is necessary to improve the stability of the antibody to improve the yield of the antibody.

Since the glycation of the antibody can affect not only the stability of the antibody but also the function in the mass production process, it is confirmed that the antibody stability can be achieved as well as the desired effect through the glycation modification of the antibody based on the glycation prediction. .

It is an object of the present invention to provide an antibody that specifically binds to clec14a, wherein the CDR of clec14a-CTLD IgG of clone 1, a clec14a-CTLD specific antibody disclosed in WO2013 / 187556, is grafted onto a commercially available therapeutic antibody , Which is substituted with a framework for therapeutic antibody, and a part of the glycosylation site in the light chain CDR1 amino acid sequence is substituted with another amino acid sequence.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating an angiogenesis-related disease comprising the antibody.

In order to solve the above-mentioned object, the present invention provides an antibody specifically binding to clec14a, which comprises a light chain variable region comprising CDR1 of TGSSSNIGXXXVT (SEQ ID NO: 1), wherein the 9th, 10th and 11th Wherein each of the amino acids X in the position is any one selected from the group consisting of R, C, G, A, T, W, S, N and V.

The present invention also provides a pharmaceutical composition for preventing or treating angiogenesis-related diseases comprising the antibody.

Other technical features and embodiments of the present invention will be more clearly described in the following detailed description and the claims that follow.

FIG. 1A is a schematic diagram showing the CDR transplantation of the parent antibody IgG into four therapeutic antibodies (adalimumab (humira), omalizumab (Xolair), tratuzumab (herceptin), bevacizumab (avastin)).
Figure 1b is a visual observation of antibody aggregation in the parent antibody IgG and clone 1 IgG preparations.
Fig. 1c is the result of aggregation index of the parent antibody IgG (green) and clone 1 IgG (orange) measured by spectrophotometry. The aggregation index was calculated as 100 x [Abs340 / (Abs280 - Abs340)].
FIG. 1D shows the quantification results of the parent antibody IgG (green) and the clone 1 IgG (orange) before and after antibody precipitation.
FIG. 2A is a schematic diagram of a desalting process for selecting four glycosylated IgG clones (deglyco C1-C4) using the phage display technique.
FIG. 2B shows the binding specificity of four kinds of desaturized IgG against hclec14a-CTLD-Fc, mclec14a-CTLD-Fc and Fc alone by ELISA.
Figure 2c shows the results of performing selection for an optimized leader antibody that inhibits clec14a mediated angiogenesis by performing an HUVEC tube formation assay. Clone 1 IgG was used as a positive control.
Figure 2d shows the results of performing an HUVEC tube formation assay to screen for an optimized leader antibody that inhibits clec14a mediated angiogenesis, wherein the total branch number is expressed as a percentage of the control (MOCK) tube formation .
Figure 2e shows the results of measuring the effect of deglyco C1 IgG on endothelial cell migration by performing a wound healing assay.
FIG. 2F shows the result of measuring the effect of deglyco C1 IgG on endothelial cell migration by performing a wound healing assay. The extent of wound difference is expressed as a percentage of the control (MOCK) migration.
Figure 2g shows the results of evaluating the mobility of deglyco C1 IgG and clone 1 IgG using one-dimensional electrophoresis under reducing conditions.
Figure 2h shows the results of evaluating the homology of deglyco C1 IgG and clone 1 IgG using two-dimensional electrophoresis.
FIG. 3A shows the results of competitive ELISA in which parent IgG binding to Deglyco C1 IgG-HRP and hclec14a-CTLD-Fc was added.
FIG. 3B shows the results of performing flow cytometry in the presence or absence (MOCK, black) of the parent antibody IgG (green) or Deglyco C1 IgG (red).
Figure 3c shows the affinity measurement of the parent antibody IgG (green) or Deglyco C1 IgG (red) for hclec14a-ECD-myc using a biolayer interferometry assay with the Octet RED96 system . * K D = equilibrium dissociation constant; K _on = association rate constant; K _off = dissociation rate constant
Figure 3d shows the results of performing HUVEC tube formation assays with or without parent antibody IgG (green), Deglyco C1 IgG (red) and bevacizumab (blue) (MOCK, black).
Figure 3E shows the results of performing HUVEC tube formation assays, wherein the total number is expressed as a percentage of the control (MOCK) tube formation.
Figure 3f shows the results of performing wound healing assays with or without parent antibody IgG (green), Deglyco C1 IgG (red) and bevacizumab (blue) (MOCK, black). Images were captured at 0 h (top) and 8 h (bottom) using a light microscope.
Figure 3g shows the results of performing a wound healing assay, wherein the extent of wound variation is expressed as a percentage of the control (MOCK) shift.
Figure 4a shows the results of counting HEK293F cells transfected with wild type clec14a and cultured for 6h under the presence or absence (MOCK, black) of the parent antibody IgG (green) or Deglyco C1 IgG (red) using a light microscope .
Figure 4b shows the number of aggregates per field as a percentage of the control (MOCK) clec14a mediated cell-cell contact.
FIG. 4C shows the results of immunohistochemical staining of clec14a-CTLD-Fc-HRP, which binds to HUVECs in the presence or absence of increasing concentrations of parent antibody IgG (green) or Deglyco C1 IgG (red) after coating HUVECs on microtiter plates The results obtained by ELISA are shown.
4d shows the results of clec14a-CTLD-Fc coated on a microtiter plate followed by clec14a-CTLD-Fc binding to clec14a-CTLD-Fc with or without increasing concentrations of either the parent antibody IgG (green) or Deglyco C1 IgG -ECD-myc as measured by ELISA.
5A shows the results of evaluating the cell viability by measuring the absorbance at 450 nm after incubation of HUVECs under absence (black) or presence (red) of deglyco C1 IgG for 2 days or in the presence of 5-FU .
Figure 5b shows the results of incubation of HUVECs in the absence (black) or presence (red) of hTNFα (pink) or deglyco C1 IgG and was detected in anti-ICAM-1 (top) or VCAM-1 (bottom) Stained and analyzed by flow cytometry. hTNF? is a positive control for endothelial cell activity.
FIG. 5c shows the results of staining of HUVECs cultured in the absence or presence of deglyco C1 IgG with rhodamine-paloidin and DAPI, and the morphology was observed with a confocal microscope.
FIG. 5d shows immunoblot analysis results for the phosphorylation of VEGF-dependent VEGFR (VEGF receptor), Akt and ERK (extracellular signal-regulated kinase) under the presence or absence of VEGF or VEGF and deglyco C1 IgG.
FIG. 5E shows the in vitro and in vivo toxicity evaluation results of the optimized lead antibody, showing the results of measurement of serum concentrations of GOT, GPT, BUN, CRE, and TBIL after 30 days of antibody injection.
FIG. 5f shows the in vitro and in vivo toxicity evaluation results of the optimized lead antibody, showing the result of measuring the mouse body weight at 1 day and 28 days after the antibody injection.
FIG. 5g shows the in vitro and in vivo toxicity evaluation results of the optimized lead antibody. The results of TUNEL assays are shown for the cell death states of kidney and liver tissues 30 days after the antibody injection.
Figure 6a shows the results of performing a VEGF-dependent tube formation assay under absence (MOCK) or presence (red) of deglyco C1 IgG.
FIG. 6B shows the results of performing a VEGF-dependent tube formation assay, wherein the total number is expressed as a percentage of the control (MOCK) tube formation.
Figure 6c shows images of VEGF-dependent vascular growth in the cut aorta rings cultured under the presence or absence (MOCK) of parent antibody IgG (green), deglyco C1 IgG (red) and bevacizumab (blue).
6D shows the results of counting the number of blood vessels grown.
Figure 6E is an image showing the formation of VEGF-dependent microvessels in Matrigel plugs in nude mice in the presence or absence (MOCK) of parent antibody IgG, Deglyco C1 IgG, and bevacizumab (MOCK).
FIG. 6F shows the result of measuring the degree of microvascular formation by measuring the hemoglobin content. The hemoglobin content is expressed as a percentage of the control (MOCK) hemoglobin content.
Figure 7a shows the results of immunohistochemistry performed with antibodies to clec14a and CD31 to detect specific expression of clec14a in blood vessels in SNU182 cancer cell xenograft mouse tissues.
FIG. 7b shows immunohistochemistry of antibodies to clec14a and CD31 to detect specific expression of clec14a in blood vessels in CFPAC-1 cancer cell xenograft mouse tissues.
FIG. 7c shows immunohistochemical staining of clec14a and CD31 antibodies to detect clec14a in tumor blood vessels of liver cancer tissues.
7d shows the results of immunohistochemical staining of clec14a and CD31 antibodies to detect clec14a in tumor blood vessels of pancreatic cancer tissues.
FIG. 7E shows the results of measuring the formation of SNU182-, CFPAC-1 or U87 cell-derived microvessels under the absence (MOCK), presence (red) or presence of bevacizumab (blue) of deglyco C1 IgG.
Figure 7f shows the results of measuring microvascular formation from SNU182-, CFPAC-1 or U87 cells, and the hemoglobin content is expressed as a percentage of the control (MOCK) hemoglobin content.
Figure 7g shows the results of measurement of microvascular formation by HCT116 and HCT116 / Beva cell derived tumors in the absence of deglyco C1 IgG and absence of bevacizumab (MOCK) or in the presence of immunoglobulin CD31.
Figure 7h shows the results of expressing CD31 positive per field as a percentage of control (MOCK) microvessel density.
Figure 7i shows the effect of deglyco C1 IgG on tumor growth, indicating that deglyco C1 IgG can reduce tumor size without affecting body weight, which is the result of measuring tumor volume and body weight once a week for one month .
Figure 8a shows the final antibody production of the produced optimized candidate antibody.
FIG. 8B shows the results of confirming the 90% purification degree and the light chain-heavy chain molecular weight of the optimized candidate antibody produced through SDS-PAGE.
Figure 9a shows the results of treating HUVEC with VEGF and an optimized antibody and confirming tube length changes.
FIG. 9B shows the result of treating HUVEC with VEGF and an optimized antibody and confirming the number of branches.
Fig. 10 is a representative picture showing the treatment of VEGF and the optimized antibody and the change of the tube length (angiogenesis progression).
FIGS. 11A and 11B show the results of analysis of the degree of tube formation after treating clone 1, 13, and 16 antibodies showing excellent anti-angiogenic activity on HUVEC cells in the presence of EGM.
FIGS. 12A and 12B show the results of analysis of inhibition of CLEC14a-mediated cell-cell contact by an optimizing antibody comprising clone 1, 13, and 16 antibodies in HEK293 cells expressing clec14a.
FIGS. 13A and 13B show results of analysis of whether the endothelial migration inhibitory effect is exhibited by the clone 1, 13 or 16 antibody.
14A is a schematic diagram of a competitive ELISA (competition ELISA) for identifying antigen binding sites of the selected antibodies.
FIG. 14B shows the results of analysis of whether or not the optimization candidate antibody binds competitively with the selected deglyco C1.
FIG. 15 shows the results of confirming whether clone 1, 13 or 16 antibodies bind to HUVEC (human umbilical vein endothelial cell) and MAEC (mouse aortic endothelial cell) cell surface using flow cytometry.
16A is a schematic diagram showing the role of CLEC14a-CTLD in vascular endothelial cell-cell junction and the mechanism by which CLEC14-CTLD-binding antibody acts as an inhibitor of cell-cell junction.
16B shows the result of confirming the role of CTLD in the binding of CLEC14a-CLEC14a.
Figure 16c shows the results of confirming whether clone 1, 13, 16 antibodies inhibited CTLD domain mediated CLEC14a molecular binding through competition ELISA.
17A is a graphical representation of CLEC14a reducing ability of vascular endothelial cell surface by CLEC14a-CTLD binding antibody.
17B shows the results of confirming the ability of the optimized antibody to reduce CLEC14a using a cell ELISA method.

In one aspect, the present invention provides an antibody specifically binding to clec14a (C-type lectin domain family 14, member A) comprising a light chain variable region comprising CDR1 of TGSSSNIGXXXVT (SEQ ID NO: 1) Wherein each of the amino acids X at the ninth, tenth and eleventh positions of the amino acid sequence of SEQ ID NO: 1 is any one selected from the group consisting of R, C, G, A, T, W, S, N and V.

The term " antibody " as used herein refers to a protein molecule comprising an immunoglobulin molecule that specifically recognizes an antigen and immunologically reacts with a specific antigen, and includes a polyclonal antibody, a monoclonal antibody, whole antibodies and antibody fragments. In addition, chimeric antibodies (eg, humanized murine antibodies), bifunctional or bispecific molecules (eg, dual specific antibodies), diabodies, triabodies and tetrabodies, Are within the scope of antibodies used in the present invention.

The whole antibody consists of two full-length light chains and two full-length heavy chains joined together as a disulfide bond between the light and heavy chains. In mammals, there are five antibody subtypes known as IgA, IgD, IgE, IgM, IgG, and IgG and are further divided into four subtypes, IgG1, IgG2, IgG3 and IgG4.

The term " antibody fragment " refers to a fragment that retains at least antigen-binding capability and may include Fab, F (ab ') 2, F (ab') 2, and Fv. The Fab consists of a variable region of each of the heavy and light chains, an invariant domain of the light chain, and a first constant domain (CH1) of the heavy chain, with the antigen binding site. Fab 'differs from Fab in that it additionally contains at least one cysteine residue at the C-terminus of the CH1 domain of the heavy chain. F (ab ') 2 consists of two Fab' molecules with disulfide bonds between the cysteine residues of the hinge region. The Fv (variable fragment) consisting of variable regions of the heavy and light chains, respectively, is a murine antibody fragment containing the original specificity of the parent immunoglobulin. Disulfide-stabilized Fv (dsFv) is formed by binding a variable region of a heavy chain to a variable region of a light chain through a disulfide bond. A short chain Fv (scFV) is an Fv covalently linked by a peptide linker at each variable region of a heavy chain and a light chain. All antibody fragments can be obtained by protease treatment of whole antibodies with proteases (e.g., papain or pepsin providing Fab or F (ab ') 2), preferably constructed by genetic recombination techniques .

The term " monoclonal antibody ", as used herein, refers to an antibody molecule having a uniform molecular composition, obtained from substantially the same group of antibodies and exhibiting binding specificity and affinity for a single epitope.

Generally, immunoglobulins have basic structural units composed of one heavy chain and two light chains. Each heavy chain is composed of one variable domain and three constant domains, while each light chain is composed of one variable domain and one constant domain. The variable region of each of the heavy and light chains comprises three complementarity-determining regions (hereinafter referred to as " CDRs ") and four framework regions. CDRs function to bind to the epitope of the antibody. The CDRs on each chain start from the N-terminus and are sequentially arranged into CDR1, CDR2 and CDR3. These are distinguished by the located chain.

The antibody according to the present invention is first characterized in that the clec14a-CTLD IgG clone 1 clec14a-CTLD human antibody (hereinafter referred to as the parent antibody) disclosed in WO2013 / 187556 filed by the inventors of the present application has a heavy chain and light chain variable region CDR1 to CDR3 sequence CDR grafting (SEQ ID NOS: 11-16), which fuses the framework regions of four FDA approved therapeutic antibodies (adalimumab (humira), omalizumab (Xolair), tratuzumab (herceptin), bevacizumab (avastin) CDR grafting).

According to one embodiment of the present invention, the degree of coagulation and the DI index (developability index) of the four antibody sequences and the parent antibody that have undergone CDR grafting were observed. The coagulation score is a numerical value that predicts the degree of coagulation in the sequence. The lower the value, the lower the cohesion. The DI index is a value that predicts the protein stability in the solution. The lower the value, the higher the solubility and stability of the antibody. As a result, while the parent antibody was predicted to have a relatively high degree of aggregation and DI index, the antibody that was substituted with omalizumab (Xolair) framework among the four antibodies had excellent aggregation degree and DI index Respectively.

Also, it was confirmed that the antibody substituted with the omalizumab (Xolair) framework was more productive than the remaining three antibodies grafted with CDR, and no aggregation occurred. In addition, it was confirmed that only the antibody substituted with the omalizumab (Xolair) framework showed cross reactivity to CTLD in humans and rats, and had similar antigenic reactivity to the parent antibody and tube formation inhibition equivalent to that of the parent antibody .

Accordingly, an antibody according to the present invention may comprise one or more heavy chain variable region frameworks selected from the group consisting of SEQ ID NOS: 17 to 20. Also, one or more light chain variable region frameworks selected from the group consisting of SEQ ID NOS: 21 to 24 can be included.

The antibody according to the present invention may be a desaturase antibody. As used herein, the term " glycosylation " refers to the presence or absence of glycosylation and the structure or form of the oligosaccharide, depending on the type of host cell, the method of manipulating the recombinant, . That is, various kinds of glycoforms may be produced depending on the sugar chain structure or the amount of sugar per constituent component of the sugar chain during the production of glycoprotein, and heterogeneity due to differences in production conditions may exist. In the case of glycoproteins having different sugar chain structures, in vivo dynamics and tissue distribution may be different from that of natural type, or they may be antagonistic to natural types, causing harmful reactions, acting as antigens for long-term continuous administration, have. Thus, oligosaccharides can be an important factor that can affect pharmacological effects and body dynamics.

As part of this oligosaccharide modulation, an antibody according to the present invention may be an antibody that has been desaturated to an antibody substituted with an omalizumab (Xolair) framework. The desaturase antibody may refer to an immunoglobulin Fc fragment that has not been glycosylated and may, for example, modify or eliminate the N-linked glycosylation site or the O-linked glycosylation site. The N-linkage may mean that the sugar chain is attached to the side chain of the asparagine residue, and the O-linkage may be one in which one of the N-acetylgalactosamine, galactose or xylose is replaced with a hydroxyamino acid, most usually serine or threonine As shown in FIG.

The modification or removal of the saccharification site may be performed by a conventional method such as a chemical method, an enzymatic method, and a genetic engineering method using a microorganism, but is not limited thereto. In a specific example according to the present invention, A random scFv library was constructed using (NNK) ₃ for the expected N-glycosylation site with the presence of one N-glycosylation site, and then a randomized scFv library was constructed using the digested display antibody Respectively.

In the present invention, the light chain variable region comprising CDR1 of TGSSSNIGXXXVT (SEQ ID NO: 1) is contained in the present invention, and the amino acids at the 9th, 10th and 11th positions of SEQ ID NO: 1 are R, C, G, T, W, S, N, and V.

In one embodiment, the amino acids at positions 9, 10 and 11 of SEQ ID NO: 1 may be RCG, ATA, WSN or AVV. CDR1 of TGSSSNIGRCGVT (SEQ ID NO: 2), CDR1 of TGSSSNIGATAVT (SEQ ID NO: 3), TGSSSNIGWSNVT (SEQ ID NO: 4) and TGSSSNIGAVVVT Lt; RTI ID = 0.0 > variable region. ≪ / RTI >

According to one embodiment of the present invention, no aggregation of the antibody was observed in the four kinds of desquered antibodies Deglyco-C1 to Deglyco-C4 selected, and it was confirmed that they had a high final purification yield. In addition, it was confirmed that the characteristics of the desaturase antibody were maintained as compared to the antibody substituted with the Xolair framework. As a result, it was confirmed that the four kinds of desquered antibodies Deglyco-C1 to Deglyco-C4 retained the cross reactivity to CTLD of human and mouse .

The amino acids at the ninth, tenth and eleventh positions of SEQ ID NO: 1 may preferably be RCG. When the amino acid at the 9th, 10th and 11th positions of SEQ ID NO: 1 is RCG, the antibody according to the present invention may comprise a light chain variable region comprising CDR1 of TGSSSNIGRCGVT (SEQ ID NO: 2).

According to one embodiment of the present invention, an antibody comprising a light chain variable region comprising CDR1 of SEQ ID NO: 2 is designated Deglyco-C1. Deglyco-C1 showed a similar degree of tube formation as the framework region of omalizumab antibody and CDR grafted antibody (clone 1 IgG) in order to confirm the efficacy of the desulfated antibody. Respectively. Further, the degree of desaturization of Deglyco-C1 was confirmed, and it was confirmed that the saccharification pattern was removed and disappeared.

A " human antibody " is a molecule consisting entirely of the amino acid sequence of all components of human immunoglobulin, including CDRs, framework regions, and the like. In the treatment of human disease, human antibodies have at least three potential advantages. First, the human antibody more preferably interacts with the human immune system to more effectively destroy the target cell, for example, by causing complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity antibody-dependent cell-mediated cytotoxicity (ADCC). Another advantage is that the human immune system does not recognize human antibodies as external molecules. Moreover, the half-life of a human antibody is similar to that occurring naturally in the human circulatory system, even when administered in smaller or less frequent doses. The antibody according to the present invention is preferably a human monoclonal antibody and has not only a strong affinity for clec14a-CTLD expressed on human endothelial cells that effectively inhibits clec14a, preferably clec14a-mediated angiogenesis Since both the heavy chain and the light chain are derived from human and have low immunogenicity, they can be useful for the treatment of angiogenesis-related diseases or cancer.

" clec14a (C-type lectin domain family 14, member A) " refers to a member of the C-type lectin / C-type lectin-like domain (CTL / CTLD) and superfamily. Clec14a is a type I transmembrane protein that is an extracellular domain that constitutes a series of epidermal growth factor-like domains C-type lectin-like domain (CTLD) and sushi-like domain transmembrane protein). Information about clec14a can be obtained from an accredited database such as NCBI GenBank. For example, human clec14a may have Gene ID No 161198, but is not limited thereto. "C-type lectin-like domain (CTLD) of clec14a (C-type lectin domain family 14, member A)" is also described as "clec14a-CTLD" or "clec14a CTLD" It means.

An " epitope " is a site that determines antigen specificity, and an antigenic determinant or an antigen determining site is used in the same sense.

Additionally, in the present invention, major amino acid residues related to the antigen-antibody reactivity of HCDR3 can be modified to improve the affinity of the desulfated antibody. For example, a randomized ab library was prepared by alanine scanning and random mutagenesis was performed to select highly reactive antibodies against the parent antibody. Each antibody can be screened for antibodies that retain characteristics and function relative to the parent antibody using a yeast surface display and a phage display technique.

In one embodiment, an antibody according to the invention may comprise a light chain variable region comprising CDR1 selected from the group consisting of SEQ ID NOS: 2-5. A light chain variable region further comprising CDR2 of SEQ ID NO: 15 and CDR3 of SEQ ID NO: 16 in said CDR1. The light chain CDR1 of SEQ ID NO: 2, the light chain CDR3 of SEQ ID NO: 15, the light chain CDR1 of SEQ ID NO: 3, the light chain CDR2 of SEQ ID NO: 15 and the light chain CDR3 of SEQ ID NO: 4, light chain CDR2 of SEQ ID NO: 15 and light chain CDR3 of SEQ ID NO: 16 or light chain CDR1 of SEQ ID NO: 5, light chain CDR2 of SEQ ID NO: 15 and light chain CDR3 of SEQ ID NO: 16.

The antibody according to the present invention may comprise a light chain variable region selected from the group consisting of SEQ ID NOS: 7 to 10. An antibody according to the present invention may comprise a light chain variable region comprising CDR1 of SEQ ID NO: 2. A light chain variable region of SEQ ID NO: 7.

In one embodiment, an antibody according to the present invention may comprise a heavy chain variable region comprising CDR3 selected from the group consisting of SEQ ID NO: 13 and SEQ ID NOs: 25-60. A heavy chain variable region further comprising CDR1 of SEQ ID NO: 11 and CDR2 of SEQ ID NO: 13 in said CDR3. The heavy chain CDR1 of SEQ ID NO: 11, the heavy chain CDR2 of SEQ ID NO: 12, the heavy chain CDR1 of SEQ ID NO: 11, the heavy chain CDR2 of SEQ ID NO: 12 and the heavy chain CDR3 of SEQ ID NO: 11 heavy chain CDR2 of SEQ ID NO: 12 and heavy chain CDR3 of SEQ ID NO: 37 or heavy chain CDR1 of SEQ ID NO: 11, heavy chain CDR2 of SEQ ID NO: 12 and heavy chain CDR3 of SEQ ID NO: 40.

A heavy chain variable region selected from the group consisting of SEQ ID NOS: 6, 61-96. An antibody according to the invention may comprise a heavy chain variable region comprising CDR3 of SEQ ID NO: 13 and / or a heavy chain variable region of SEQ ID NO:

The light chain CDR1 of SEQ ID NO: 2, the light chain CDR2 of SEQ ID NO: 15, the light chain CDR3 of SEQ ID NO: 11, the heavy chain CDR2 of SEQ ID NO: 12, and the heavy chain CDR3 of SEQ ID NO: 13;

Light chain CDR1 of SEQ ID NO: 3, light chain CDR2 of SEQ ID NO: 15 and light chain CDR3 of SEQ ID NO: 16, heavy chain CDR1 of SEQ ID NO: 11, heavy chain CDR2 of SEQ ID NO: 12 and heavy chain CDR3 of SEQ ID NO: 13;

A light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 15, a light chain CDR3 of SEQ ID NO: 11, a heavy chain CDR2 of SEQ ID NO: 12, and a heavy chain CDR3 of SEQ ID NO: 13;

Light chain CDR1 of SEQ ID NO: 5, light chain CDR2 of SEQ ID NO: 15 and light chain CDR3 of SEQ ID NO: 16, heavy chain CDR1 of SEQ ID NO: 11, heavy chain CDR2 of SEQ ID NO: 12 and heavy chain CDR3 of SEQ ID NO: 13;

The light chain CDR1 of SEQ ID NO: 2, the light chain CDR2 of SEQ ID NO: 15, and the light chain CDR3 of SEQ ID NO: 11, the heavy chain CDR2 of SEQ ID NO: 12 and the heavy chain CDR3 of SEQ ID NO:

The light chain CDR1 of SEQ ID NO: 2, the light chain CDR2 of SEQ ID NO: 15 and the light chain CDR3 of SEQ ID NO: 16, the heavy chain CDR1 of SEQ ID NO: 11, the heavy chain CDR2 of SEQ ID NO: 12 and the heavy chain CDR3 of SEQ ID NO:

The light chain CDR1 of SEQ ID NO: 2, the light chain CDR2 of SEQ ID NO: 15 and the light chain CDR3 of SEQ ID NO: 16, the heavy chain CDR1 of SEQ ID NO: 11, the heavy chain CDR2 of SEQ ID NO: 12 and the heavy chain CDR3 of SEQ ID NO:

An antibody according to the present invention comprises a light chain variable region of SEQ ID NO: 7 and a heavy chain variable region of SEQ ID NO: 6;

A light chain variable region of SEQ ID NO: 8 and a heavy chain variable region of SEQ ID NO: 6;

A light chain variable region of SEQ ID NO: 9 and a heavy chain variable region of SEQ ID NO: 6;

A light chain variable region of SEQ ID NO: 10 and a heavy chain variable region of SEQ ID NO: 6;

A light chain variable region of SEQ ID NO: 7 and a heavy chain variable region of SEQ ID NO: 61;

A light chain variable region of SEQ ID NO: 7 and a heavy chain variable region of SEQ ID NO: 73; or

A light chain variable region of SEQ ID NO: 7, and a heavy chain variable region of SEQ ID NO: 76.

IgG, IgA, IgD, IgE, IgM, or combinations or hybrids thereof, when the antibody of the present invention comprises a constant domain.

As used herein, " combination " means that a polypeptide encoding a short-chain immunoglobulin Fc fragment of the same origin is linked to a short-chain polypeptide of another origin to produce a dimer or multimer. This indicates that the dimer or multimer may be formed from two or more constant domains selected from the group consisting of the constant domains of IgG, IgA, IgD, IgE and IgM.

As used herein, the term " hybrid " means that sequences encoding two or more heavy chain constant domains of different origin are present in the short chain immunoglobulin heavy chain constant domain. For example, the domain hybrid may be composed of one to four domains selected from the group consisting of CH1, CH2, CH3 and CH4 of IgG, IgA, IgD, IgE and IgM. In addition, the combination of hybrids can be generated from the heavy chain constant domains of IgG1, IgG2, IgG3 and IgG4, the subtypes of IgG. The combination of the hybrids is as defined above.

In addition, the antibody of the present invention may further comprise a light chain constant region, and may be derived from a lambda (貫) or kappa (kappa) light chain.

Preferably, the antibody may be a human monoclonal antibody capable of specifically inhibiting angiogenesis by specifically binding to human clec14a-CTLD as well as murine and clec14a-CTLD. The ability of human antibodies to function in both humans and mice, in other words cross-reactivity, provides the advantage that human antibodies enable preclinical studies in mice.

In another aspect, the present invention relates to a composition for inhibiting angiogenesis, comprising the antibody, or a pharmaceutical composition for preventing or treating angiogenesis-related diseases.

Since the antibody according to the present invention can effectively inhibit angiogenesis, a composition comprising the antibody as an effective ingredient is also useful for inhibiting angiogenesis, and further useful for preventing or treating angiogenesis-related diseases can do.

As used herein, the term " suppression of angiogenesis " refers to inhibiting the formation or growth of new blood vessels from previously existing ducts. For the purposes of the present invention, inhibition of angiogenesis may be achieved by inhibiting cell migration, intercellular contact, more preferably clec14a-mediated cell migration, clec14a-mediated intercellular contact, HUVEC migration or tube formation, clec14a CLTD-CLTD complex Lt; / RTI >

As used herein, the term " angiogenesis-related disease " refers to a disease associated with the onset or progression of angiogenesis. If the antibody can be treated, the disease can be included in a range of angiogenesis-related diseases without limitation. Examples of angiogenesis related diseases include cancer, metastasis, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, macular degeneration, angiogenesis Neovascular glaucoma, erythrosis, proliferative retinopathy, psoriasis, hemophilic arthritis, capillary formation of atherosclerotic plaques, keloids, , Wound granulation, vascular adhesion, rheumatoid arthritis, osteoarthritis, autoimmune diseases, Crohn's disease, restenosis, Atherosclerosis, intestinal adhesions, cat scratch disease, ulcer, liver cirrhosis, nephritis, diabetic kidney One comprising a ring (diabetic nephropathy), diabetes mellitus (diabetes mellitus), inflammatory diseases (inflammatory diseases) and neurodegenerative disorders (neurodegenerative diseases), but is not limited to such. In addition, the cancer may be an esophageal cancer, a stomach cancer, a large intestine cancer, a rectal cancer, an oral cancer, a pharynx cancer, a larynx cancer, (Eg, lung cancer, colon cancer, breast cancer, uterine cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer testicular cancer, bladder cancer, renal cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, But are not limited to, brain cancer, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma, and multiple myeloid blood cancer. It is not.

As used herein, the term " prevention or prophylaxis " refers to any action which, by administering an antibody or composition of the invention, inhibits or delays the onset of a disease of interest. The term " treatment or therapy " refers to any action that results in administration of an antibody or composition of the invention to ameliorate or ameliorate symptoms of a disease of interest.

The composition comprising the antibody of the invention is preferably a pharmaceutical composition and may further comprise suitable vehicles, excipients or diluents typically used in the art.

A pharmaceutical composition comprising a pharmaceutically acceptable vehicle may be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, nonaqueous solutions, suspensions, lyophilizates, It may be in a variety of oral or parenteral dosage forms. In this context, the pharmaceutical composition of the present invention may be formulated in combination with diluents or excipients such as fillers, thickeners, binders, humectants, disintegrants, surfactants and the like. Solid dosage forms for oral administration may be in the form of tablets, pills, powders, granules, capsules, and the like. With respect to these solid suspensions, the compounds of the present invention may be formulated in combination with one or more excipients such as starch, calcium carbonate, sucrose, lactose, or gelatin. Lubricants such as simple excipients, magnesium stearate, talc, and the like may be further used. Liquid preparations for oral administration may be suspensions, solutions, emulsions, syrups, and the like. Various excipients such as water or liquid paraffin, simple diluents, wetting agents, sweeteners, aromatics, preservatives and the like may be included in the liquid preparation. In addition, the pharmaceutical composition of the present invention may be in a parenteral dosage form such as a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilizate, a suppository, or the like. Injectable propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and esters such as ethyl oleate may be suitable for non-aqueous solvents and suspensions. The basic materials of suppositories include Witepsol, macrogol, Tween 61, cacao butter, laurin butter and glycerogelatin.

The composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term " pharmaceutically effective amount " refers to the amount of a pharmaceutical composition for treating a disease sufficient for a reasonable benefit and risk ratio applicable to all medical treatments. The effective amount will depend on the severity of the disease to be treated, the age and sex of the patient, the type of disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration, It depends on various factors including parameters. The compositions of the present invention may be administered alone or in combination with other therapies. In such a case, they may be administered sequentially or simultaneously with conventional therapies. In addition, the composition may be administered in a single dose or in divided doses. When fully considering these factors, it is important to administer a minimal amount sufficient to achieve maximum efficacy without adverse effects, and this dose can be readily determined by an expert in the field. The dosage of the pharmaceutical composition of the present invention is not particularly limited, but may be varied depending on various factors including the health condition and the weight of the patient, the severity of the disease, the kind of the medicine, the administration route and the administration time. The compositions may be administered orally, rectally, rectally, intravenously, subcutaneously, orally in a mammalian, including, but not limited to, rats, mice, , Intrauterinely, or intracerebrovascularly. ≪ / RTI >

In another aspect, the present invention relates to a method for inhibiting angiogenesis, a method for preventing or treating angiogenesis-related diseases comprising the step of administering the antibody or the composition to a subject in need thereof.

The antibody, composition and angiogenesis inhibition are as described above.

In detail, the method of inhibition of the present invention comprises the step of administering to a subject in need of inhibition of angiogenesis an amount of the pharmaceutical composition in a pharmaceutically effective amount. The subject may be, but is not limited to, mammals such as dogs, cows, horses, rabbits, mice, rats, chickens and humans. The pharmaceutical compositions may be administered parenterally, subcutaneously, intraperitoneally, intrapulmonarily or intranasally, and if necessary, intralesional administration for topical treatment. ≪ / RTI > The preferred dose of the pharmaceutical composition of the present invention will vary depending on various factors including the health condition and weight of the individual, the severity of the disease, the kind of the drug, the administration route and the administration time, and can be easily determined by those skilled in the art.

In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating cancer comprising the antibody, or a method for preventing or treating cancer, comprising administering the antibody or the composition to a subject in need thereof. The terms " antibody ", " prevention ", and " treatment "

If it is treatable with an antibody of the present invention, cancer is not limited. Cancers that develop clec14a-mediated tumor progression are preferred. In detail, the antibody of the present invention can prevent the development or progression of cancer by inhibiting angiogenesis. Examples of cancer include esophageal cancer, stomach cancer, large intestine cancer, rectal cancer, oral cancer, pharynx cancer, larynx cancer, lung cancer, lung cancer, colon cancer, breast cancer, uterine cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, testicular cancer, bladder cancer, renal cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, But are not limited to, brain cancer, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma, and multiple myeloid blood cancer.

In addition, the antibodies of the present invention may be used in combination with other antibodies or biologically active agents or materials for various purposes.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is apparent to those skilled in the art that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: Fabrication of stability improving antibodies by grafting in silico- based CDRs

WO2013 / 187556 has reported that clec14a-CTLD parent antibody can specifically regulate angiogenesis in vitro . However, in the case of the parent antibody (clone 1 of WO2013 / 187556), it was confirmed that aggregation occurred remarkably in the purification process, and further optimization process for improving the yield of antibody was required. Therefore, in silico based CDR grafting was performed. Six CDRs (Table 1) in the heavy and light chain variable regions of the parent antibody were assigned to the frameworks of each of the FDA approved therapeutic antibodies (adalimumab (humira), omalizumab (Xolair), tratuzumab (herceptin), and bevacizumab (Fig. 1A).

In silico- based analysis and Discovery studio 3.1 software provided by Accelrys, the DI index (developability index) of the four antibodies (clone 1 to 4 IgG) and CDR grafted antibody was compared. DI is a rapid in silico predictor used to evaluate monoclonal antibodies according to their aggregation characteristics. The higher the DI, the higher the degree of cohesion.

As shown in Table 2, clone 1 IgG having the framework region of omalizumab (Xolair) (the specific sequence of the framework region is shown in Table 3) among the four antibodies that carried out graft and CDR grafting (IgG of clone 2-4) that had undergone antibody and CDR grafting, which means that the stability of clone 1 IgG is excellent.

To confirm the improved cohesion of clone 1 IgG, the agglutination of purified parent antibody IgG and clone 1 IgG was visualized and then confirmed by spectrophotometry. Aggregation was observed only in the parent antibody IgG and not in the clone 1 IgG (Fig. 1B). The aggregation indices of the parent antibody IgG and the clone 1 IgG measured by spectrophotometry were about 23.4 and 1.5, respectively, which means that the clone 1 IgG is more soluble than the parent antibody IgG (FIG. 1C).

To further compare the stability of the parent antibody IgG and clone 1 IgG, the aggregates were precipitated and removed by centrifugation, and the soluble antibody was quantitated. Approximately 95% of the antibodies in the parent antibody IgG preparation were aggregated whereas no aggregation of antibody was observed in the clone 1 IgG (FIG. 1d). These results suggest that in silico- based CDR grafting is an effective method for the production of antibody platforms with improved stability.

Example 2: Selection of optimized antibodies by continuous desulfurization process and functional separation

Glycation heterogeneity in therapeutic proteins can have a significant impact on the quality characteristics of biopharmaceuticals during development and production. In silico- based clone 1 IgG, the putative N-glycosylation sites in the light chain CDR1 of the clone 1 IgG were identified through glycation assays. In order to eliminate such expected N-glycosylation sites, a semisynthetic antibody library having random mutations at the N-glycosylation site was prepared and a desolvation process was performed. Specifically, a semisynthetic scFv (single chain variable fragment) randomized with NNK trinucleotide oligonucleotides was prepared and successively bio-panned using phage display technology including magnetic bead-coated human and mouse clec14a-CTLD Respectively. Finally, after sequencing the DNA of the selected scFv clones, four clones that strongly reacted to both human and mouse clec14a-CTLDs (hclec14a-CTLD and mclec14a-CTLD) and different amino acid sequences at the predicted N- (Fig. 2A).

The scFv clone was then converted to IgG, expressed in HEK (human embryonic kidney) 293 cells, and then purified. According to the ELISA analysis, all purified desaturase IgG (deglyco C1-C4 IgGs) specifically binds to both hclec14a-CTLD-Fc and mclec14a-CTLD-Fc, but not to Fc alone, clec14a-CTLD-specific antibody (Fig. 2B).

In addition, as shown in Table 4, no aggregation was observed in the four kinds of desaturized IgG (deglyco C1-C4 IgGs), and deglyco C1 and deglyco C2 showed high final purification yields.

The genetic variation introduced in antibody engineering induces unexpected defects and consequently reduces antibody function. In order to isolate antibodies that inhibit clec14a mediated angiogenesis, tube formation and endothelial migration assays using HUVECs (human umbilical vein endothelial cells) were performed in the presence or absence of clone 1 IgG and deglyco C1-C4 IgG. Deglyco C1 IgG was selected as the lead antibody among four deglyco IgGs because deglyco C1 IgG showed the best inhibitory effect on HUVEC tube formation (Fig. 2c and Fig. 2d) and migration (Fig. 2e and 2f). In addition, it was confirmed that deglyco C1 IgG contained the desired change in the amino acid sequence (N32R / N33C / S34G) in the light chain CDR1.

To analyze the biochemical properties of Deglyco C1 IgG, the mobility of deglyco C1 IgG and clone 1 IgG was first evaluated using 1D electrophoresis under reducing conditions. The molecular weight of the Deglyco C1 VL was 25 kDa, similar to the expected molecular weight, while the molecular weight of the clone 1 VL, the pre-degradation form, was higher (Fig. 2g).

Two-dimensional electrophoresis also shows that a heterologous pattern of clone 1 scFv present in the lower range of the isoelectric point (pI) is not present in the deglyco C1 scFv, indicating enhanced homology of deglyco C1 IgG (Fig. 2h).

Overall, these results suggest that the selected leading antibody effectively inhibits pathological angiogenesis and is an anti-angiogenic antibody with improved homogeneity.

Example 3: Biochemical and functional properties of optimized leader antibodies

To determine the clec14a-CTLD binding site of Deglyco C1 IgG, deglyco C1 IgG conjugated with horseradish peroxidase (HRP) (deglyco C1 IgG-HRP) was prepared and then subjected to competitive ELISA.

The deglyco C1 IgG-HRP binding to hclec14a-CTLD-Fc was exceptionally competitive by addition of parent antibody IgG, which may indicate that the parent antibody and deglyco C1 have similar clec14a-CTLD binding sites (Fig. 3a).

To determine if Deglyco C1 IgG binds to human endothelial cells, flow cytometry analysis was performed with HUVECs. Deglyco C1 IgG specifically bound to the surface of HUVECs in a manner similar to the parent antibody IgG. In order to measure the binding affinity of deglyco C1 IgG to hclec14a-ECD (extracellular domain of human clec14a), real-time measurement of antibody-antigen binding was performed, and deglyco C1 IgG was used to measure the binding affinity of about 6 nM KD (equilibrium dissociation constant) constant (Fig. 3C).

Overall, these results show that, despite the two-step optimization process, the binding specificity of the optimized leader antibody remains similar to that of the parent antibody IgG.

In order to compare the inhibitory effects of Deglyco C1 IgG and bevacizumab in vitro on angiogenesis, tube formation and migration assays were performed in the presence or absence of bevacizumab as the parent antibody IgG, deglyco C1 IgG or as a positive control . Deglyco C1 IgG significantly inhibited HUVEC tube formation (Figs. 3d and 3e) and inhibited migration to a similar degree to bevacizumab in vitro (Fig. 3f and 3g). This means that the anti-angiogenic activity of the lead antibody corresponds to bevacizumab.

Example 4: New mechanism of action of optimized leader antibody

To confirm the mechanism of action of deglyco C1 IgG in angiogenesis, the number of cell aggregates formed through HEK293F cells transfected with wild type clec14a and cultured in the presence or absence of the parent antibody IgG or deglyco C1 IgG was monitored and clec14a mediated cells - A functional assay for cell contact was performed.

The parent antibody IgG was used as a positive control to specifically block clec14a mediated cell-cell contact. Deglyco C1 IgG was shown to inhibit the aggregation of cells transfected with wild-type clec14a in a manner similar to that of the parent antibody IgG, suggesting the inhibitory effect of deglyco C1 IgG on clec14a mediated cell-cell contact in angiogenesis 4a and 4b).

CTLD-Fc-HRP (HRP-labeled hclec14a-CTLD-Fc-HRP) with or without increasing concentrations of the parent antibody IgG or deglyco C1 IgG in order to analyze the deglyco C1 IgG action mechanism specifically in angiogenesis at the molecular level. Lt; / RTI > were treated with HUVECs. Deglyco C1 IgG significantly inhibited clec14a-CTLD binding to HUVECs in a concentration-dependent manner similar to the parent antibody IgG (Figure 4c).

In addition, hclec14a-CTLD-Fc-HRP was incubated with purified hclec14a-ECD with or without increasing concentrations of parent antibody IgG or deglyco C1 IgG. deglyco C1 IgG directly inhibited the molecular interactions between hclec14-CTLD and hclec14a-ECD in a concentration-dependent manner similar to the parent antibody IgG (Fig. 4d).

Overall, these results suggest that the antibody produced directly blocks the molecular interaction between clec14a-CTLD mediated clec14a molecules and acts as an interactive blocker to specifically inhibit endothelial cell-cell contact during angiogenesis.

Example 5: Effect of optimized lead antibodies on endothelial cell toxicity and VEGF signaling in endothelial cells

To assess the effect of deglyco C1 IgG on endothelial cell toxicity, the viability of HUVECs was measured after deglyco C1 IgG treatment. Survival was measured according to the manufacturer's manual through Cell Counting Kit-8 (# CK04-13, Dojindo Laboratories, Kumamoto, Japan).

5-FU (5-fluorouracil) significantly reduced the viability of HUVECs, whereas HUVECs did not show cytotoxic effects on these cells (Fig. 5A).

Immunocytochemistry was also used to assess the morphology of HUVEC in the presence or absence of deglyco C1 IgG. Deglyco C1 IgG did not alter the morphology of HUVEC (Fig. 5B). HUVECs were treated with deglyco C1 IgG and treated with VCAM-1 (vascular cell adhesion molecule-1) and ICAM-1 (intercellular cell adhesion) in order to evaluate the effect of deglyco C1 IgG on the endothelial cell activity, HUVEC activity was monitored by measuring the expression of endothelial cell activation markers containing the HUVEC-molecule-1. Human tumor necrosis factor (hTNFa) was used as a positive control for endothelial cell activity. deglyco C1 IgG had little effect on HUVEC activity, whereas hTNFa induced HUVEC activity as expected (Figure 5c).

To determine the effect of deglyco C1 IgG on endothelial VEGF-dependent signaling, VEGF-treated HUVECs were immunoblotted in the presence or absence of deglyco C1 IgG to detect VEGFR (VEGF receptor), Akt and extracellular signal-regulated kinase (ERK) Was monitored for phosphorylation. Deglyco C1 IgG had little effect on VEGF-dependent phosphorylation of VEGFR, Akt and ERK in HUVECs (Fig. 5d).

In order to confirm the in vivo toxicity of deglyco C1 IgG, normal mice were intravenously injected with deglyco C1 IgG twice a week, and liver and kidney function, body weight, and apoptosis status of liver and kidney tissues were compared between the control and experimental groups. Liver function was determined by measuring the serum concentrations of glutamic-oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), and total bilirubin (TBIL). Renal function measured BUN (blood urea nitrogen) and creatinine Respectively. Cell death was measured by TUNEL staining.

As a result, there was no significant change in liver function and kidney function (Fig. 5d). There was no significant change in body weight (FIG. 5E). In addition, a cell death state was observed (Fig. 5F). These results suggest that deglyco C1 IgG does not cause severe in vivo toxicity.

Overall, these results suggest that deglyco C1 IgG does not cause severe endothelial cell toxicity or negatively affect VEGF mediated signaling in normal endothelial cells in vivo .

Example 6: Effect of optimized lead antibody on VEGF-dependent angiogenesis

To confirm the in vitro effect of Deglyco C1 IgG on VEGF-dependent angiogenesis, HUVEC tube formation assays were performed after treatment of HUVECs with VEGF in the presence or absence of deglyco C1 IgG. 150 [mu] l of Matrigel was placed in a 48 well plate and incubated at 37 [deg.] C for 30 min. HUVECs (10 ⁵ ) cultured in EGM-2 were harvested and inoculated onto Matrigel-coated plates and incubated for 18h at 37 ° C in the presence or absence of clone 1 IgG, parent antibody IgG, depleted clec14a-CTLD IgG or bevacizumab Lt; / RTI > HUVECs cultured in VEGF-containing endothelial cell basal medium were inoculated onto Matrigel coated plates and incubated for 18 hours at 37 ° C in the presence or absence of deglyco C1 IgG (20 μg ml ^-1 ).

Deglyco C1 IgG eliminated VEGF-dependent tube formation at a significantly near perfect level (Figure 6a).

An ex vivo rat aortic ring assay was performed in the presence or absence of deglyco C1 IgG and bevacizumab to further confirm the effect of deglyco C1 IgG on VEGF-dependent angiogenesis. In the rat aorta, blood vessels hardly grow to EBM (endothelial basal medium), but most of the blood vessels in the VEGF expression condition grew in the rat aorta. Furthermore, it was observed that deglyco C1 IgG reduced the number of blood vessels growing in the rat aorta by a similar effect to bevacizumab when incubated with VEGF (Figs. 6b and 6c).

In order to observe the in vivo efficacy of deglyco C1 IgG in VEGF-dependent angiogenesis, a mouse Matrigel model was established and the hemoglobin level was measured as a marker of microvascularization, and the amount of microvessels in the mouse Matrigel plug assay The inhibitory effects of deglyco C1 IgG and bevacizumab on formation were compared. Deglyco C1 IgG significantly reduced hemoglobin content in a manner similar to bevacizumab (Fig. 6d and Fig. 6e).

Overall, these results suggest that the antibodies produced may be effective in inhibiting VEGF-dependent abnormal angiogenesis in vivo .

Example 7: Effect of optimized lead antibody on tumor angiogenesis

In order to confirm the association of clec14a in tumor angiogenesis, xenograft tumor models including SNU182 human hepatocyte or CFPAC-1 human pancreatic cancer cell carcinoma were first established, and clec14a and the known endothelial marker protein CD31 Immunohistochemistry was performed via commercially available antibodies. Immunohistochemistry HUVECs (5 x 10 ⁴ ) grown in 0.1% (w / v) gelatin coated glass cover slips were incubated at 37 ° C for 24 hours in the presence or absence of deglyco C1 IgG. Cells were fixed with 4% (w / v) PFA and blocked with PBS containing 5% (w / v) BSA and 0.1% (v / v) Triton X-100 at 37 ° C for 1 hour, - Paloidin (1 unit / well) and Hoechst 33258 dye for 1 hour.

Similar to CD31, clec14a was expressed in blood vessels of SNU182- and CFPAC-1 tumor xenografts, which implies that clec14a is specifically expressed in tumor vessels (Figs. 7a and 7b).

To evaluate the specific expression of clec14a in tumor blood vessels in clinical samples using immunohistochemistry, we compared the expression of clec14a in normal and hepatic and pancreatic cancer tissues. As a result, it was confirmed that clec14a was significantly and specifically increased in the blood vessels of the cancer patient tissue, but not in the normal blood vessels (FIGS. 7C and 7D).

To confirm the effect of deglyco C1 IgG on tumor angiogenesis in vivo , a tumor cell-derived Matrigel plug angiogenesis assay was performed in thymus-removed nude mice. SNU182-, CFPAC-1 cells and U87 human glioma cells containing Matrigel were transplanted into thymus-free nude mice in the presence or absence of 10 mg / kg single dose deglyco C1 IgG and bevacizumab. Two weeks later, Matrigel plugs were removed and the total hemoglobin content in each group was measured with an ELISA reader. Deglyco C1 IgG significantly reduced SNU182-, CFPAC-1 and U87 human glioma-derived hemoglobin to a similar degree as bevacizumab (Figs. 7e and 7f).

For U87 human glioma, deglyco C1 IgG also significantly reduced the tumor size of U87 cells as well as bevacizumab without affecting body weight (Fig. 7i). It was confirmed that deglyco C1 IgG did not significantly affect survival, shape or activation of HUVEC in vitro . This means that the optimized antibody does not cause significant endotoxic toxicity in vivo .

Using HCT116 human colon cancer cells and HCT116 human colorectal cancer cells (HCT116 and HCT116 / Beva) with bevacizumab in the presence or absence of a single dose of deglyco C1 IgG or bevacizumab 5 mg / kg, Matrigel plug angiogenesis Respectively. After 14 days the matrigel plugs were removed and immunohistochemistry was performed on CD31 and microvascular markers. Microvessel density was determined by quantifying CD31 positivity per field in each of the images obtained by confocal microscopy. deglyco C1 IgG or bevacizumab HCT116 cells to a similarly significant degree. Similarly, microvascular formation by HCT116 / Beva cells was significantly inhibited by deglyco C1 IgG, but not by bevacizumab (Fig. 7g, Fig. 7h). This demonstrates the superior ability of deglyco C1 IgG to inhibit tumor angiogenesis in bevacizumab resistant colorectal cancer cells.

Collectively, these results suggest that the antibody produced has the ability to inhibit tumor angiogenesis in vivo .

Example 8: Selection and efficacy analysis of optimized lead antibody

Example 8-1. Screening of Optimizing Lead Antibodies

In order to screen additional optimizing antibodies, 36 antibodies were further selected and antibodies with improved affinity compared to deglyco C1 were screened. Mammalian expression vectors containing 36 antibodies (IgG antibody form) in Table 5 were transfected into HEK293 cells using PEI (polyethylenimine) and cultured for 40 days in 7 days. The culture was obtained by centrifugation and then purified through affinity column chromatography with protein A sepharose column. A purification degree of 90% was confirmed by SDS-PAGE, and the molecular weights of the light chain and the heavy chain were simultaneously confirmed (FIG. 8A).

Opti 1, Opti 3 and Opti 16 (clones 1, 3 and 16), which are highly productive clones (about 50 mg / L or more), were selected as primary cultures.

Example 8-2. In vitro efficacy of selected antibodies

Optimization of VEGF-dependent angiogenesis for major angiogenesis factor In order to confirm the angiogenesis inhibitory ability of the leading antibody, use IncuCyte FLR live content imaging system (Essen Bioscience Inc) capable of real-time angiogenesis analysis, Angiogenesis were compared and analyzed. First, GFP-transfected HUVECs were plated on 94-well plates and the in vitro efficacy was compared by treating 36 antibodies at a concentration of 20 ug / ml. At this time, the efficacy with the optimized antibody was compared and analyzed using parental IgG (parent antibody: clone 1 of WO2013 / 187556), deglyco C1 and bevacizumab (avastin) as positive control.

The results are shown in FIGS. 9A and 9B, respectively. It was confirmed that the antibodies treated with both the tube length (FIG. 9A) and the branch point (FIG. 9B) had anti-angiogenic activity, The strong potency of clones 1, 3 and 16 was confirmed.

As shown in FIG. 10 showing representative results of treatment of VEGF and the optimized antibody and confirming the change of the tube length (angiogenesis progression), it was confirmed that the tube length was prolonged (angiogenesis progression) human IgG, control antibody) was found to be non-responsive as a negative control for the administered antibody. Suramin (angiogenesis inhibitor, small chemical) and avastin (IgG) were also used as positive control. In particular, it was confirmed that the optimized antibody clones 1, 3 and 16 had the same efficacy as the positive control groups suramin and avastin.

EGM has been used to induce strong angiogenesis using vascular endothelial cells as a complex of various angiogenic factors. Therefore, in order to confirm whether anti-angiogenesis exists even in the presence of various angiogenic factors such as EGM as well as VEGF-dependent angiogenesis of the optimized antibody, HUVEC cells were cultured in a 48-well microtiter plate in the presence of EGM After clone 1, 13, and 16, which showed excellent anti-angiogenic activity, were treated at a concentration of 20 μg / ml, respectively, and the degree of tube formation was measured using the original antibody C1 (original C1) and deglyco C1 as a positive control Respectively.

The results are shown in FIGS. 11A and 11B, and finally confirmed that the selected antibodies significantly inhibited EGM-dependent angiogenesis. Thus, it was confirmed that the optimized antibodies including the clone 1, 13, and 16 antibodies can effectively inhibit angiogenesis caused by various angiogenic factors.

The major mechanism of angiogenesis is understood as the main mechanism of endothelial cell-cell contact in addition to tube length, numbers of branches and tube formation described above. Therefore, HEK293 cells were transfected with a mammalian expression vector containing clec14a in order to proceed with CLEC14a-mediated cell-cell contact, and the cell-cell contact (aggregate, clec14a As a marker of mediating cell junction). Aggregate was manually counted under a microscope to compare the efficacy of the antibody to the antibody. To confirm the anti - angiogenic activity of the treated antibody, 20 ug / ml of each antibody was treated with a parent antibody (original C1) and deglyco C1, respectively, as a positive control. The results are shown in Figs. 12A and 12B, and finally it was confirmed that all the antibodies excellently inhibited CLEC14a-mediated cell-cell contact.

In order to analyze the effectiveness of the optimized leading antibody to move, one of the other functions of the angiogenic endothelium (endothelial migration) in vitro standing, IncuCyte FLR live content imaging system (Essen Bioscience Inc) to use the HUVEC migration assay (HUVEC migration assay. At this time, wounds were uniformly wounded using a wound maker provided by Incucyte, and treated with 20 ug / ml of antibody to evaluate migration inhibition. The results are shown in FIGS. 13A and 13B and final confirmation that the optimized antibodies including clones 1, 13, and 16 had the same endothelial migration inhibition as the parent antibody (original C1), deglyco C1 IgG, and avastin Respectively.

Example 8-3. Identification of antigen binding sites of selected antibodies

Competitive ELISA (competition ELISA) was performed to identify antigen binding sites of the selected antibodies. Specifically, the CTLD antigen was coated on a 96-well microtiter plate and induced binding with HRP-conjugated deglyco C1 IgG (FIG. 14A). At the same time, each of the 36 antibodies was treated and confirmed to bind to the antigen competitively with deglyco C1, thereby confirming the antigen binding sites of deglyco C1 IgG and 36 species of antibody. The results are shown in FIG. 14B, and it was confirmed that all 36 kinds of antibodies had antigen binding sites similar to deglyco C1 IgG.

Example 8-4. Identification of interspecific cross-reactivity of optimized antibodies

Human umbilical vein endothelial cells (HUVECs) and mouse aortic endothelial cells (MAECs) were cultured to confirm cross-species reactivity of the optimized antibodies. Twenty cells / ml of original antibody (C1) deglyco C1, and clones 1, 13, and 16, respectively, to confirm the ability of binding to the two cell surfaces was confirmed by flow cytometry. The results are shown in Fig. In conclusion, the three clones 1, 13, and 16 of the optimized antibodies showed interspecies cross-reactivity that could bind to human and murine CLEC14a.

Example 8-5. Mechanism of action of optimized antibody

In order to analyze the mechanism of action of the optimized antibodies, the inhibitory effect of the optimized antibody on the cell - cell junction of vascular endothelial cells was first confirmed. The inventors of the present application have found that the CTLD domain of CLEC14a plays an important role in vascular endothelial cell-cell junction and the CLEC14-CTLD-binding antibody (original Cl) plays a role as an interaction inhibitor thereof ).

To prove this, COS-7 cells were transfected with wild-type CLEC14a and CLEC14a DCTLD lacking the CTLD domain, respectively, and their cell lysates were coated on a 96-well microtiter plate. In addition, purified HRP-conjugated CTLD-Fc was incubated to confirm CTLD binding. The results are shown in Fig. 16B. In conclusion, it was confirmed that the CTLD domain is important for CLEC14a-CLEC14a binding. Figure 16c shows the results of comparative analysis of the degree of binding of HRP-conjugated CTLD-Fc to CLEC14a while increasing the molar ratio of antigen to antibody by 0: 1, 1: 1, 1: 2 through competition ELISA, The original antibody C1 (original C1) and deglyco C1 were used as positive control, respectively, and three kinds of optimized antibody representative clones 1, 13 and 16 were treated as a control group. As a result, it was confirmed that all the treatment antibodies significantly inhibited the CTLD-mediated interaction between CTLD-mediated interaction between CTEC domain-mediated interaction between CLEC14a molecules.

In addition, down-regulation of CLEC14a on vascular endothelial cell surface was confirmed for the mechanism of action of the optimized antibodies. 17A is a graphical representation of CLEC14a reducing ability of vascular endothelial cell surface by a conventional CLEC14a-CTLD binding antibody (parent antibody: original Cl). HUVECs were coated on 96-well microtiter plates to confirm CLEC14a abilities of the optimized antibodies and treated with 20 ug / ml parent antibody (original Cl), deglyco C1, and three clone 1, 13, 16 optimized antibodies The quantitative level of CLEC14a present on the surface of the hourly HUVEC was finally confirmed using a cell ELISA (cell ELISA) method. The quantitative level of CLEC14a was determined using commercially available sheep anti-CLEC14a antibody (sheep anti-CLEC14a Ab) and HRP-conjugated anti-human antibody with secondary antibody, followed by TMB And the absorbance was confirmed at 450 nm using an ELISA reader. The results are shown in Fig. 17B, and it was confirmed that the treated antibody showed CLEC14a reduction ability on the vascular endothelial cell surface.

The desaturase antibody specifically binding to clec14a according to the present invention exhibits excellent yield, maintains cross-reactivity with humans and rats, exhibits the desired antigen reactivity, exhibits excellent solubility and stability of the antibody due to less aggregation As well as an improved antibody capable of exhibiting improved antibody properties and efficacy with improved tube formation inhibitory ability.

While the present invention has been described in detail with reference to specific embodiments thereof, it is to be understood by those skilled in the art that such description is directed to preferred embodiments and that the scope of the invention is not limited. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

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<110> SCRIPPS KOREA ANTIBODY INSTITUTE <120> Deglycobilted Antibody Binding Details to clec14a and Uses          Thereof <130> PP-B1950 <150> KR 10-2016-0115577 <151> 2016-09-08 <160> 96 <170> Kopatentin 2.0 <210> 1 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> L-CDR1 <400> 1 Thr Gly Ser Ser Ser Asn Ile Gly Xaa Xaa Xaa Val Thr   1 5 10 <210> 2 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> L-CDR1 <400> 2 Thr Gly Ser Ser Ser Asn Ile Gly Arg Cys Gly Val Thr   1 5 10 <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> L-CDR1 <400> 3 Thr Gly Ser Ser Ser Asn Ile Gly Ala Thr Ala Val Thr   1 5 10 <210> 4 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> L-CDR1 <400> 4 Thr Gly Ser Ser Ser Asn Ile Gly Trp Ser Asn Val Thr   1 5 10 <210> 5 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> L-CDR1 <400> 5 Thr Gly Ser Ser Ser Asn Ile Gly Ala Val Val Val Thr   1 5 10 <210> 6 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Deg C1 VH <400> 6 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Thr Trp Trp Val Leu Gly Pro Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 7 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Deg C1 VL <400> 7 Asp Ile Gln Leu Thr Gln Ser Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Gly Ser Ser Ser Asn Ile Gly Arg              20 25 30 Cys Gly Val Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu          35 40 45 Leu Ile Tyr Ala Asp Ser His Arg Pro Ser Gly Val Ser Ser Arg Phe      50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu  65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Ala Trp Asp Asp Ser                  85 90 95 Leu Ser Gly Tyr Val Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg             100 105 110 <210> 8 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Deg C2 VL <400> 8 Asp Ile Gln Leu Thr Gln Ser Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala              20 25 30 Thr Ala Val Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu          35 40 45 Leu Ile Tyr Ala Asp Ser His Arg Pro Ser Gly Val Ser Ser Arg Phe      50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu  65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Ala Trp Asp Asp Ser                  85 90 95 Leu Ser Gly Tyr Val Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg             100 105 110 <210> 9 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Deg C3 VL <400> 9 Asp Ile Gln Leu Thr Gln Ser Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Gly Ser Ser Ser Asn Ile Gly Trp              20 25 30 Ser Asn Val Thr Trp Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu          35 40 45 Leu Ile Tyr Ala Asp Ser His Arg Pro Ser Gly Val Ser Ser Arg Phe      50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu  65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Ala Trp Asp Asp Ser                  85 90 95 Leu Ser Gly Tyr Val Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg             100 105 110 <210> 10 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Deg C4 VL <400> 10 Asp Ile Gln Leu Thr Gln Ser Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala              20 25 30 Val Val Val Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu          35 40 45 Leu Ile Tyr Ala Asp Ser His Arg Pro Ser Gly Val Ser Ser Arg Phe      50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu  65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Ala Trp Asp Asp Ser                  85 90 95 Leu Ser Gly Tyr Val Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg             100 105 110 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> H-CDR1 <400> 11 Gly Phe Thr Phe Ser Gly Tyr Asp Met Ser   1 5 10 <210> 12 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> H-CDR2 <400> 12 Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 13 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 13 Gly Ala Thr Trp Trp Val Leu Gly Pro Phe Asp Tyr   1 5 10 <210> 14 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> L-CDR1 <400> 14 Thr Gly Ser Ser Ser Asn Ile Gly Asn Asn Ser Val Thr   1 5 10 <210> 15 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> L-CDR2 <400> 15 Ala Asp Ser His Arg Pro Ser   1 5 <210> 16 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> L-CDR3 <400> 16 Gly Ala Trp Asp Asp Ser Leu Ser Gly Tyr Val   1 5 10 <210> 17 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> VH FR1 <400> 17 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser              20 25 <210> 18 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> VH FR2 <400> 18 Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala   1 5 10 <210> 19 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> VH FR3 <400> 19 Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr Leu Gln   1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg              20 25 30 <210> 20 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VH FR4 <400> 20 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser   1 5 10 <210> 21 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> VL FR1 <400> 21 Asp Ile Gln Leu Thr Gln Ser Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys              20 <210> 22 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> VL FR2 <400> 22 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr   1 5 10 15 <210> 23 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> VL FR3 <400> 23 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr   1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys              20 25 30 <210> 24 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL FR4 <400> 24 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg   1 5 10 <210> 25 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 25 Gly Ala Ala Thr Lys Val Leu Gly Lys Phe Asp Cys   1 5 10 <210> 26 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 26 Gly Ala Ala Ser Pro Val Leu Gly Pro Phe Asp Glu   1 5 10 <210> 27 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 27 Gly Ala Thr Trp Trp Val Leu Gly Ala Phe Asp Tyr   1 5 10 <210> 28 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 28 Gly Ala Glu Phe Val Val Leu Gly Ser Phe Asp Met   1 5 10 <210> 29 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 29 Gly Ala Ala Arg Cys Val Leu Gly Asn Phe Asp His   1 5 10 <210> 30 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 30 Gly Ala Ser Arg Thr Val Leu Gly Asn Phe Asp Asp   1 5 10 <210> 31 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 31 Gly Ala His Leu Thr Val Leu Gly Ile Phe Asp Thr   1 5 10 <210> 32 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 32 Gly Ala His Ile Tyr Val Leu Gly Gln Phe Asp Gly   1 5 10 <210> 33 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 33 Gly Ala Asn Leu Gly Val Leu Gly Glu Phe Asp Lys   1 5 10 <210> 34 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 34 Gly Ala Val Pro Pro Val Leu Gly Ala Phe Asp Thr   1 5 10 <210> 35 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 35 Gly Ala Arg Ile Asp Val Leu Gly Pro Phe Asp Ala   1 5 10 <210> 36 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 36 Gly Ala Lys Pro Pro Val Leu Gly Gln Phe Asp Arg   1 5 10 <210> 37 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 37 Gly Ala Met Glu Gln Val Leu Gly Pro Phe Asp Leu   1 5 10 <210> 38 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 38 Gly Ala Arg Ser Trp Val Leu Gly Glu Phe Asp Lys   1 5 10 <210> 39 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 39 Gly Ala Glu Cys Ala Val Leu Gly Asn Phe Asp Tyr   1 5 10 <210> 40 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 40 Gly Ala Arg Arg Gln Val Leu Gly Cys Phe Asp Phe   1 5 10 <210> 41 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 41 Gly Ala Ser Ala Pro Val Leu Gly Asp Phe Asp Tyr   1 5 10 <210> 42 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 42 Gly Ala Ser Thr Pro Val Leu Gly Ser Phe Asp Asn   1 5 10 <210> 43 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 43 Gly Ala His Tyr Asn Val Leu Gly Pro Phe Asp Ser   1 5 10 <210> 44 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 44 Gly Ala Arg Thr Cys Val Leu Gly Pro Phe Asp His   1 5 10 <210> 45 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 45 Gly Ala Ile Ala Pro Val Leu Gly Gly Phe Asp Arg   1 5 10 <210> 46 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 46 Gly Ala Arg Gln Ser Val Leu Gly Pro Phe Asp Ala   1 5 10 <210> 47 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 47 Gly Ala His Tyr Asn Val Leu Gly Pro Phe Asp Ser   1 5 10 <210> 48 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 48 Gly Ala Lys Tyr Tyr Val Leu Gly His Phe Asp Glu   1 5 10 <210> 49 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 49 Gly Ala Ala Leu Arg Val Leu Gly Met Phe Asp Leu   1 5 10 <210> 50 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 50 Gly Ala His Arg Arg Val Leu Gly Trp Phe Asp Asp   1 5 10 <210> 51 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 51 Gly Ala Tyr His Thr Val Leu Gly Gln Phe Asp Gln   1 5 10 <210> 52 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 52 Gly Ala Gln Ser Thr Val Leu Gly Asn Phe Asp Thr   1 5 10 <210> 53 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 53 Gly Ala His Met Arg Val Leu Gly Pro Phe Asp Gly   1 5 10 <210> 54 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 54 Gly Ala Ala Leu His Val Leu Gly Pro Phe Asp Pro   1 5 10 <210> 55 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 55 Gly Ala His Gly Gln Val Leu Gly Asp Phe Asp Gln   1 5 10 <210> 56 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 56 Gly Ala Ser Gln Glu Val Leu Gly Asp Phe Asp Val   1 5 10 <210> 57 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 57 Ser Ala Thr Trp Trp Met Ser Glu Pro Arg Ser Tyr   1 5 10 <210> 58 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 58 Gly Ala Asp Asn His Val Leu Gly Asp Phe Asp Val   1 5 10 <210> 59 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 59 Gly Ala His Lys Pro Val Leu Gly Asp Phe Asp Ala   1 5 10 <210> 60 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 60 Gly Ala His His Ala Val Leu Gly Pro Phe Asp Arg   1 5 10 <210> 61 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 61 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Ala Thr Lys Val Leu Gly Lys Phe Asp Cys Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 62 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 62 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Ala Ser Pro Val Leu Gly Pro Phe Asp Glu Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 63 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 63 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Thr Trp Trp Val Leu Gly Ala Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 64 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 64 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Glu Phe Val Val Leu Gly Ser Phe Asp Met Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 65 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 65 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Ala Arg Cys Val Leu Gly Asn Phe Asp His Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 66 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 66 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Ser Arg Thr Val Leu Gly Asn Phe Asp Asp Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 67 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 67 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala His Leu Thr Val Leu Gly Ile Phe Asp Thr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 68 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 68 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala His Ile Tyr Val Leu Gly Gln Phe Asp Gly Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 69 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 69 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Asn Leu Gly Val Leu Gly Glu Phe Asp Lys Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 70 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 70 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Val Pro Pro Val Leu Gly Ala Phe Asp Thr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 71 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 71 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Arg Ile Asp Val Leu Gly Pro Phe Asp Ala Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 72 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 72 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Lys Pro Pro Val Leu Gly Gln Phe Asp Arg Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 73 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 73 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Met Glu Gln Val Leu Gly Pro Phe Asp Leu Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 74 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 74 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Arg Ser Trp Val Leu Gly Glu Phe Asp Lys Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 75 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 75 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Glu Cys Ala Val Leu Gly Asn Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 76 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 76 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Arg Arg Gln Val Leu Gly Cys Phe Asp Phe Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 77 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 77 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Ser Ala Pro Val Leu Gly Asp Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 78 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 78 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Phe Phe Cys                  85 90 95 Ala Arg Gly Ala Ser Thr Pro Val Leu Gly Ser Phe Asp Asn Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 79 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 79 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Phe Tyr Cys                  85 90 95 Ala Arg Gly Ala His Tyr Asn Val Leu Gly Pro Phe Asp Ser Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 80 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 80 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Arg Thr Cys Val Leu Gly Pro Phe Asp His Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 81 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 81 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Ile Ala Pro Val Leu Gly Gly Phe Asp Arg Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 82 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 82 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Arg Gln Ser Val Leu Gly Pro Phe Asp Ala Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 83 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 83 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala His Tyr Asn Val Leu Gly Pro Phe Asp Ser Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 84 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 84 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Lys Tyr Tyr Val Leu Gly His Phe Asp Glu Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 85 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 85 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Ala Leu Arg Val Leu Gly Met Phe Asp Leu Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 86 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 86 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala His Arg Arg Val Leu Gly Trp Phe Asp Asp Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 87 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 87 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Tyr His Thr Val Leu Gly Gln Phe Asp Gln Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 88 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 88 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Gln Ser Thr Val Leu Gly Asn Phe Asp Thr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 89 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 89 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala His Met Arg Val Leu Gly Pro Phe Asp Gly Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 90 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 90 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Ala Leu His Val Leu Gly Pro Phe Asp Pro Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 91 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 91 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala His Gly Gln Val Leu Gly Asp Phe Asp Gln Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 92 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 92 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Ser Gln Glu Val Leu Gly Asp Phe Asp Val Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 93 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 93 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Ser Ala Thr Trp Trp Met Ser Glu Pro Arg Ser Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 94 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 94 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Asp Asn His Val Leu Gly Asp Phe Asp Val Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 95 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 95 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala His Lys Pro Val Leu Gly Asp Phe Asp Ala Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 96 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 96 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Gly Tyr              20 25 30 Asp Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Tyr Pro Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala His His Ala Val Leu Gly Pro Phe Asp Arg Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120

Claims (12)

As an antibody specifically binding to clec14a,
A light chain variable region comprising CDR1 of TGSSSNIGXXXVT (SEQ ID NO: 1)
Wherein each of the amino acids X at the ninth, tenth and eleventh positions of SEQ ID NO: 1 is any one selected from the group consisting of R, C, G, A, T, W, S, N and V. The method according to claim 1,
Wherein the amino acids at positions 9, 10 and 11 of SEQ ID NO: 1 are RCG, ATA, WSN or AVV. The antibody of claim 1, wherein the antibody is desaturated. The method according to claim 1,
Wherein the antibody comprises a light chain variable region comprising at least one framework region selected from the group consisting of SEQ ID NOS: 21-24. The method according to claim 1,
Wherein the antibody comprises a light chain variable region selected from the group consisting of SEQ ID NOS: 7 to 10. The method according to claim 1,
A heavy chain variable region comprising CDR3 selected from the group consisting of SEQ ID NO: 13, SEQ ID NOs: 25-60. The method according to claim 1,
A heavy chain variable region selected from the group consisting of SEQ ID NOS: 6, 61-96. The method according to claim 1,
The light chain CDR1 of SEQ ID NO: 2, the light chain CDR2 of SEQ ID NO: 15 and the light chain CDR3 of SEQ ID NO: 16, the heavy chain CDR1 of SEQ ID NO: 11, the heavy chain CDR2 of SEQ ID NO: 12 and the heavy chain CDR3 of SEQ ID NO: 13;
Light chain CDR1 of SEQ ID NO: 3, light chain CDR2 of SEQ ID NO: 15 and light chain CDR3 of SEQ ID NO: 16, heavy chain CDR1 of SEQ ID NO: 11, heavy chain CDR2 of SEQ ID NO: 12 and heavy chain CDR3 of SEQ ID NO: 13;
A light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 15, a light chain CDR3 of SEQ ID NO: 11, a heavy chain CDR2 of SEQ ID NO: 12, and a heavy chain CDR3 of SEQ ID NO: 13;
Light chain CDR1 of SEQ ID NO: 5, light chain CDR2 of SEQ ID NO: 15 and light chain CDR3 of SEQ ID NO: 16, heavy chain CDR1 of SEQ ID NO: 11, heavy chain CDR2 of SEQ ID NO: 12 and heavy chain CDR3 of SEQ ID NO: 13;
The light chain CDR1 of SEQ ID NO: 2, the light chain CDR2 of SEQ ID NO: 15, and the light chain CDR3 of SEQ ID NO: 11, the heavy chain CDR2 of SEQ ID NO: 12 and the heavy chain CDR3 of SEQ ID NO:
The light chain CDR1 of SEQ ID NO: 2, the light chain CDR2 of SEQ ID NO: 15 and the light chain CDR3 of SEQ ID NO: 16, the heavy chain CDR1 of SEQ ID NO: 11, the heavy chain CDR2 of SEQ ID NO: 12 and the heavy chain CDR3 of SEQ ID NO:
The light chain CDR1 of SEQ ID NO: 2, the light chain CDR2 of SEQ ID NO: 15 and the light chain CDR3 of SEQ ID NO: 16, the heavy chain CDR1 of SEQ ID NO: 11, the heavy chain CDR2 of SEQ ID NO: 12 and the heavy chain CDR3 of SEQ ID NO: 40. The method according to claim 1,
A light chain variable region of SEQ ID NO: 7 and a heavy chain variable region of SEQ ID NO: 6;
A light chain variable region of SEQ ID NO: 8 and a heavy chain variable region of SEQ ID NO: 6;
A light chain variable region of SEQ ID NO: 9 and a heavy chain variable region of SEQ ID NO: 6;
A light chain variable region of SEQ ID NO: 10 and a heavy chain variable region of SEQ ID NO: 6;
A light chain variable region of SEQ ID NO: 7 and a heavy chain variable region of SEQ ID NO: 61;
A light chain variable region of SEQ ID NO: 7 and a heavy chain variable region of SEQ ID NO: 73; or
A light chain variable region of SEQ ID NO: 7 and a heavy chain variable region of SEQ ID NO: 76. 9. A pharmaceutical composition for preventing or treating angiogenesis-related diseases comprising the antibody of any one of claims 1 to 9. 11. The method of claim 10,
The angiogenesis related diseases are selected from the group consisting of cancer, metastasis, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, macular degeneration, angiogenesis glaucoma neovascular glaucoma, erythrosis, proliferative retinopathy, psoriasis, hemophilic arthritis, capillary formation of atherosclerotic plaques, keloid, But are not limited to, wound granulation, vascular adhesion, rheumatoid arthritis, osteoarthritis, autoimmune diseases, Crohn's disease, restenosis, Atherosclerosis, intestinal adhesions, cat scratch disease, ulcer, liver cirrhosis, nephritis, diabetic nephropathy, diabetic nephropathy, (Diabetic nephropathy), diabetes mellitus (diabetes mellitus), inflammatory diseases (inflammatory diseases) and neurodegenerative disorders (neurodegenerative diseases) a composition according to claim, selected from the group consisting of. 12. The method of claim 11,
The cancer may be an esophageal cancer, a stomach cancer, a large intestine cancer, a rectal cancer, an oral cancer, a pharynx cancer, a larynx cancer, a lung cancer lung cancer, colon cancer, breast cancer, uterine cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testis cancer, Cancer, bladder cancer, renal cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain tumor wherein the composition is selected from the group consisting of brain cancer, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma and multiple myeloid blood cancer.

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